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Plant transformation without selection

a plant and transformation technology, applied in the field of plant biotechnology, can solve the problems of time-consuming and inefficient systems, complicated subsequent analyses and product development efforts, and inability to select plants,

Active Publication Date: 2008-03-06
MONSANTO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008] In one aspect, the invention provides a method for identifying transgenic corn plants, comprising: (a) obtaining corn plant cells transformed with a DNA segment comprising a nucleic acid sequence of interest; (b) regenerating a plurality of corn plants or differentiated corn plant parts from the cells without first selecting for the presence of said DNA segment; and (c) identifying at least a first transgenic corn plant or differentiated plant part from the plurality of corn plants or differentiated corn plant parts. In some embodiments, the DNA segment does not comprise a selectable marker gene, or a visual marker gene. In other embodiments, the plants are regenerated by growth on solid media, liquid media, or a combination of solid and liquid media. In particular embodiments, the plants are regenerated by growth solely on liquid media subsequent to contacting the cells with a GOI and prior to identifying the transgenic corn plant or transgenic differentiated plant part. In certain embodiments, the transformation frequency of cells grown solely in liquid media subsequent to contacting the cells with a GOI and prior to identification of transgenic plants or transgenic plant parts is enhanced relative to the transformation frequency observed when cells are grown in solid media or soil subsequent to contacting the cells with a GOI and prior to identification of transgenic plants or transgenic plant parts.

Problems solved by technology

However, upon creating a transformed plant comprising a GOI, a selectable or screenable marker gene which is not itself a GOI is typically no longer necessary, and its presence may complicate subsequent analyses and product development efforts.
Furthermore, the necessity of a strong promoter to drive a selectable marker has been shown to bias the expression of the desired gene (Yoo et al., 2005).
However most of these systems are time-consuming and inefficient.

Method used

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Examples

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example 1

Marker-Free Transformation

[0068] Transformation of regenerable immature corn embryos may be performed via a Rhizobia-mediated protocol, e.g. as generally described by Cai et al. (U.S. Patent Application Publication 20040244075). In particular, a modified Agrobacterium-mediated method was used. Immature embryos with a size range of 1.9-2.5 mm, for instance about 2.3 mm, were selected from corn ears and co-cultivated with an ABI Agrobacterium strain C58 to mediate the transfer of DNA into the plant cells containing the recombinant construct of interest, for instance pMON93040 containing both GUS and CP4 EPSPS under the expression control of an actin promoter, to allow for both visual analysis of transformed cells and sectors, and to allow for use of a Weathermax™ glyphosate spray as a surrogate for a later, post-regeneration screen followed by a confirmation test by a PCR-based screen for transformed plants. Larger embryos, e.g. about 2.5 mm or up to about 3.2 mm in size, may also be...

example 2

Efficient Development of Transgenic Sectors without Selection Pressure during Callus Proliferation

[0069] A system for efficient regeneration of transgenic plants in the absence of a selection agent was developed. Following co-culture of an explant with Agrobacterium (4 days on Lynx 1898 medium (Table 1), callus proliferation commenced on Lynx 1316 (Table 1), for 10-14 days, without selection. Next, pre-regeneration of callus tissue was performed for 10 days on Lynx 1844 medium (Table 1), followed by regeneration on Lynx 1344 (Table 1) for 10 days, and Lynx 1471 for 3 weeks (Table 1). All steps except for culture on Lynx 1471 were performed without use of a selective agent; thus callus growth and plant regeneration occurred without a selective agent for about 4 weeks after co-cultivation. Growth of regenerating plants in the last step, on Lynx 1471, was performed in the presence of a low level of glyphosate (0.02 mM, v / v) to estimate the maximum possible transformation frequency. Pr...

example 3

Additional Corn Transformation and Regeneration Experiments, and Screening of Putative Transformed Plants

[0071] Three more studies were performed to confirm that efficient regeneration of transgenic sectors was routinely possible without applying selection at any stage. The plasmid used was pMON93040, described above. Following co-culture on Lynx 1898 for 1 day, callus proliferation was performed on Lynx 1316 for 10 days, pre-regeneration on Lynx 1844 for 10 days, and regeneration on Lynx 1344 for 3 days, followed by growth on Lynx 1607. A single corn ear was used to isolate the embryos for each experiment, and the embryos ranged in size from 2.8-3.2 mm. In two of the studies, embryo inoculation was performed directly isolating embryos into Agrobacterium suspension at O.D.660=1.0, while in the other study embryos were first isolated into 1 ml of liquid Lynx 1013 medium (1 Liter: MS Basal Salts (Phytotech): 2.165 g; MS Vitamins (100×; Phytotech): 10 ml; Sucrose (Phytotech): 68.5 g; ...

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Abstract

The invention provides methods for identifying regenerated transformed plants and differentiated transformed plant parts, obtained without subjecting plant cells to selective conditions prior to regenerating the cells to obtain differentiated tissues. In particular embodiments, the plant cells are corn plant cells. Methods for growing and handling plants, including identifying plants that demonstrate specific traits of interest are also provided.

Description

[0001] This application claims the priority of U.S. provisional application Ser. No. 60 / 841,519 filed Aug. 31, 2006, the entire disclosure of which is incorporated herein by reference.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The invention relates to the field of plant biotechnology. In particular, the invention relates to methods for producing transgenic plants not requiring use of a selectable marker gene prior to obtaining a regenerated plant or plant part. [0004] 2. Description of the Related Art [0005] Stable transformation of plant cells and production of transgenic plants has typically required a selection step, wherein plant tissue is selected in the presence of a selection agent after having been contacted by one or more exogenous nucleic acid sequences, including ones that comprise a sequence or sequences encoding a gene of interest and a marker gene. Following such selection, stably transformed plants comprising a gene of interest (GOI) may be re...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12N15/8201C12N15/8205C12N15/8209C12N15/8206C12N15/8207C12N15/8265C12Q2600/158C12Q1/6876C12Q2600/13C12N15/8202C12N15/821C12Q1/6895C12Q1/6827C12Q1/686Y02A40/146C12N15/8212A01H6/4684C12N15/8261C12Q1/6813C12Q1/25G01N33/53A01H1/04A01H4/001
Inventor ROUT, JYOTI R.
Owner MONSANTO TECH LLC
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